CN109090697B - Method for fermenting tobacco by mixed bacteria - Google Patents
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- CN109090697B CN109090697B CN201810798971.3A CN201810798971A CN109090697B CN 109090697 B CN109090697 B CN 109090697B CN 201810798971 A CN201810798971 A CN 201810798971A CN 109090697 B CN109090697 B CN 109090697B
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- A—HUMAN NECESSITIES
- A24—TOBACCO; CIGARS; CIGARETTES; SIMULATED SMOKING DEVICES; SMOKERS' REQUISITES
- A24B—MANUFACTURE OR PREPARATION OF TOBACCO FOR SMOKING OR CHEWING; TOBACCO; SNUFF
- A24B15/00—Chemical features or treatment of tobacco; Tobacco substitutes, e.g. in liquid form
- A24B15/18—Treatment of tobacco products or tobacco substitutes
- A24B15/20—Biochemical treatment
Abstract
The invention discloses a method for fermenting tobacco by mixed bacteria, which comprises the following steps: inoculating strains of Pichia pastoris FBKL2.0008 and Issatchenkia orientalis FBKL2.0002 on a slant culture medium, culturing at 28 ℃ for 24-48 h, and allowing the bacteria to grow over a test tube; respectively streaking and activating the slant culture strains on a malt extract agar culture medium, and culturing at 28 deg.C for 24-48 h; preparing a seed solution: mixing the cultured yeast seed liquid of the pichia pastoris and the seed liquid of the issatchenkia orientalis to obtain a mixed bacterial liquid, wherein the pichia pastoris seed liquid accounts for 60-80% of the mixed bacterial liquid; adding the mixed bacterial liquid into the tobacco according to 0.4ml/g, supplementing sterile water 0.2ml/g tobacco every 3d, and culturing at 28-30 ℃ for 15-20 d. Drying at 40-60 deg.C for 15-30 min. The invention has the fragrance of rose flower, powder and honey, has the wine fragrance, simple process, difficult pollution and wide application of products.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for fermenting tobacco by issatchenkia orientalis and pichia pastoris.
Background
After the tobacco leaves are harvested, the tobacco leaves are processed in different modes according to the characteristics and the processing tradition of the tobacco leaves, primarily saccharified, matured and further processed. The tobacco leaves are fermented to change the chemical components of the tobacco, so that the flavor becomes softer and the content of nicotine in the tobacco leaves is reduced. The novel processing technology of tobacco turns to the research of tobacco flavor from the improvement of planting technology, the current report uses the culture solution prepared by actinorhizobium separated from tobacco leaves to carry out the root irrigation and the leaf surface spraying treatment of the tobacco, and the result proves that the NNN content is obviously reduced in the later airing period, thereby reducing the TSNA content; microbial strains for degrading nicotine are separated from tobacco leaf surfaces and tobacco planting soil, strains with higher activity are obtained after primary screening and secondary screening, and the upper tobacco leaves are treated by adopting crude enzyme liquid of the strains, so that the results show that impure gas and irritation can be obviously relieved by enzyme liquid treatment, and the smoking quality is obviously improved; four strains of the bacillus subtilis separated from the tobacco are inoculated to the tobacco leaves in the storage period by adopting single bacteria or mixed bacteria, so that the fragrance of the tobacco leaves is obviously improved; adding Aspergillus oryzae, fermenting, and making into tobacco shred; the quality of tobacco shreds can be improved by adding starch syrup, fermentation promoting peptide, etc., and the artificial fermentation of cigar coat is realized by adding flos Chrysanthemi, green tea, and rice wine to improve flavor and quality.
Cigarettes with different style characteristics of white spirit are popular, and a method for enhancing flavor by directly adding white spirit is generally adopted. No report is available on a method for preparing tobacco with liquor style characteristics by directly fermenting tobacco by adopting liquor-producing aroma-producing yeast.
Disclosure of Invention
The invention aims to overcome the defects and provide the method for fermenting the tobacco, which has the fragrance like rose flower fragrance, powder fragrance and honey, has the wine fragrance, is simple in process, is not easy to pollute and has wide product application.
The invention relates to a method for fermenting tobacco by mixed bacteria, which comprises the following steps:
(1) slant culture: inoculating Pichia pastoris FBKL2.0008(Pichia kudriavzevii FBKL2.0008) and Issatchenkia orientalis FBKL2.0002(Issatchenkia orientalis) strains on a slant culture medium, culturing at 28 ℃ for 24-48 h, and allowing the strains to grow into test tubes;
(2) activating strains: respectively streaking and activating the slant culture strains on a malt extract agar culture medium, and culturing at 28 deg.C for 24-48 h;
(3) preparing a seed solution: preparing a liquid culture medium, sterilizing at the temperature of 115 ℃ and 121 ℃ for 20min, then respectively inoculating activated strains into the liquid culture medium on a superclean bench, and culturing for 24-48 h on a shaking table at the temperature of 28 ℃;
(4) preparing mixed bacterial liquid: mixing the cultured yeast seed liquid of the pichia pastoris and the seed liquid of the issatchenkia orientalis to obtain a mixed bacterial liquid, wherein the pichia pastoris seed liquid accounts for 60-80% of the mixed bacterial liquid;
(5) tobacco fermentation: adding the mixed bacterial liquid into the tobacco according to 0.4ml/g, supplementing sterile water 0.2ml/g tobacco every 3d, and culturing at 28-30 ℃ for 15-20 d. Drying at 40-60 deg.C for 15-30 min.
The method for fermenting tobacco by mixed bacteria comprises the following steps: and (3) in the tobacco fermentation step in the step (5), the tobacco can be cut into threads before being fermented, and can also be fermented after being cut into threads to obtain a finished product.
The method for fermenting tobacco by mixed bacteria comprises the following steps: the slant culture medium is malt extract agar culture medium, and comprises 130g/L malt extract powder, 0.1g/L chloramphenicol, 15g/L agar, and pH of 5.8-6.2.
The method for fermenting tobacco by mixed bacteria comprises the following steps: pichia pastoris FBKL2.0008(Pichia kudriavzevii FBKL2.0008) was deposited at 17.6.2015 to the China center for type culture Collection (address: university of Wuhan, China), with the following deposition numbers: CCTCC NO: m2015380.
The method for fermenting tobacco by mixed bacteria comprises the following steps: issatchenkia orientalis FBKL2.0002(Issatchenkia orientalis FBKL2.0002) was deposited at 35.07.2018 in the China center for type culture Collection (address: university of Wuhan, China), with a deposit number: CCTCC NO: m2018251.
Compared with the prior art, the invention has obvious beneficial effects, and the technical scheme can show that: the pichia pastoris in the white spirit fermentation has stronger capacity of producing the phenethyl acetate, the phenethyl acetate has rose fragrance, powder fragrance and honey fragrance, is similar to apple fragrance and cocoa and whisky fragrance, the highly diluted and weak phenethyl acetate fragrance has the functions of calming, calming and hypnosis, the issatchenkia orientalis has stronger capacity of producing the wine, the content of the ethanol can reach more than 60 percent of volatile matters at most, and the characteristics can obviously enhance the fragrance, improve the taste and cover the offensive odor, and bring pleasant feeling to people. The Issatchenkia orientalis in the white spirit fermentation has stronger capacity of producing ethanol, and also produces isoamyl alcohol, alpha-thujene and other fragrant substances. The fermented tobacco prepared by solid fermentation of mixed bacteria of Pichia pastoris FBKL2.0008 and Issatchenkia orientalis FBKL2.0002 has safety on component analysis through GC-MS, and has high yield of aromatic alcohol, outstanding fragrance and no obvious stimulation in the fermentation process. The tobacco leaves are fermented by utilizing the strains which highly produce the phenethyl acetate and the ethanol in the white spirit brewing process, the obtained product releases pleasant smell in the burning process of the cigarettes, and the product is from natural fermentation and has the characteristics of no pollution, no toxicity and the like. The tobacco prepared by the invention has the fragrance of rose flower, powder fragrance and honey, and also has the fragrance of wine, the process is simple, the pollution is not easy, the requirement on the production environment is not high, and the produced tobacco can be processed into cigarettes, and can also be used as oral tobacco products, snuff and the like.
Detailed Description
Example 1:
a method for fermenting tobacco by mixed bacteria comprises the following steps:
(1) slant culture: inoculating Pichia pastoris FBKL2.0008(Pichia kudriavzevii FBKL2.0008) and Issatchenkia orientalis FBKL2.0002(Issatchenkia orientalis FBKL2.0002) strains on a slant culture medium, culturing at 28 ℃ for 48h, and allowing the strains to grow full of test tubes;
(2) activating strains: respectively streaking and activating the slant culture strains on a malt extract agar culture medium, and culturing at 28 ℃ for 24 h;
(3) preparing a seed solution: preparing liquid culture medium (malt extract powder 130g/L, chloramphenicol 0.1g/L, pH5.8-6.2), placing in a triangular flask, sterilizing at 121 deg.C for 20min, inoculating activated strains into the liquid culture medium on a clean bench, and culturing on a shaker at 28 deg.C for 48 hr;
(4) preparing mixed bacterial liquid: mixing the cultured yeast seed liquid of the pichia pastoris and the seed liquid of the issatchenkia orientalis to obtain a mixed bacterial liquid, wherein the pichia pastoris seed liquid accounts for 80 percent of the mixed bacterial liquid; .
(5) Tobacco fermentation: adding the mixed bacterial liquid into the tobacco according to 0.4ml/g, supplementing sterile water 0.2ml/g tobacco every 3d, and culturing in an environment at 30 ℃ for 15 d. Drying at 40 deg.C for 30min, and cutting the fermented tobacco into tobacco shreds.
Example 2:
a method for fermenting tobacco by mixed bacteria comprises the following steps:
(1) slant culture: inoculating strains of Pichia pastoris FBKL2.0008 and Issatchenkia orientalis FBKL2.0002 on a slant culture medium, and culturing at 28 ℃ for 35h until the strains grow full of a test tube;
(2) activating strains: respectively streaking and activating the slant culture strains on a malt extract agar culture medium, and culturing at 28 ℃ for 30 h;
(3) preparing a seed solution: preparing liquid culture medium (malt extract powder 130g/L, chloramphenicol 0.1g/L, pH5.8-6.2), placing in a triangular flask, sterilizing at 121 deg.C for 20min, inoculating activated strains into the liquid culture medium on a clean bench, and culturing on a shaker at 28 deg.C for 38 h;
(4) preparing mixed bacterial liquid: mixing the cultured yeast seed liquid of the pichia pastoris and the seed liquid of the issatchenkia orientalis to obtain a mixed bacterial liquid, wherein the pichia pastoris seed liquid accounts for 70 percent of the mixed bacterial liquid;
(5) tobacco fermentation: adding the mixed bacterial liquid into tobacco shreds at a ratio of 0.4ml/g, supplementing sterile water 0.2ml/g tobacco every 3d, culturing at 30 deg.C for 20d, and drying at 60 deg.C for 15 min.
Example 3
A method for fermenting tobacco by mixed bacteria comprises the following steps:
(1) slant culture: inoculating Pichia pastoris FBKL2.0008(Pichia kudriavzevii) and Issatchenkia orientalis FBKL2.0002(Issatchenkia orientalis) strains on a slant culture medium, and culturing at 28 ℃ for 24h until the strains grow full of a test tube;
(2) activating strains: respectively streaking and activating the slant culture strains on a malt extract agar culture medium, and culturing at 28 ℃ for 24 h;
(3) preparing a seed solution: preparing liquid culture medium (malt extract powder 130g/L, chloramphenicol 0.1g/L, pH5.8-6.2), placing in a triangular flask, sterilizing at 121 deg.C for 20min, inoculating activated strains into the liquid culture medium on a clean bench, and culturing on a shaker at 28 deg.C for 24 hr;
(4) preparing mixed bacterial liquid: mixing the cultured yeast seed liquid of the pichia pastoris and the seed liquid of the issatchenkia orientalis to obtain a mixed bacterial liquid, wherein the pichia pastoris seed liquid accounts for 60 percent of the mixed bacterial liquid; .
(5) Tobacco fermentation: adding the mixed bacterial liquid into tobacco shreds at a ratio of 0.4ml/g, adding sterile water 0.2ml/g tobacco every 3 days, culturing at 18 deg.C for 15 days, and drying at 50 deg.C for 20 min. And (4) carrying out subsequent processing and packaging on the tobacco shreds obtained by fermentation to obtain finished products.
The obtained product has nicotine content and volatile components before and after fermentation
Reference table for scoring and quantitative description of individual indexes of sensory quality of single cigarette
Gray: 7.0-9.0 is white, 6.0-6.9 is white gray, 4.0-5.9 is off-white, 3.0-3.9 is gray black, and 0-2.9 is black.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, and any simple modification, equivalent change and modification made to the above embodiment according to the technical spirit of the present invention are within the scope of the present invention without departing from the technical spirit of the present invention.
Claims (5)
1. A method for fermenting tobacco by mixed bacteria comprises the following steps:
(1) slant culture: inoculating Pichia pastoris FBKL2.0008(Pichia kudriavzevii FBKL2.0008) and Issatchenkia orientalis FBKL2.0002(Issatchenkia orientalis) strains on a slant culture medium, culturing at 28 ℃ for 24-48 h, and allowing the strains to grow into test tubes;
(2) activating strains: respectively streaking and activating the slant culture strains on a malt extract agar culture medium, and culturing at 28 deg.C for 24-48 h;
(3) preparing a seed solution: preparing a liquid culture medium, sterilizing at the temperature of 115 ℃ and 121 ℃ for 20min, then respectively inoculating activated strains into the liquid culture medium on a superclean bench, and culturing for 24-48 h on a shaking table at the temperature of 28 ℃;
(4) preparing mixed bacterial liquid: mixing the cultured yeast seed liquid of the pichia pastoris and the seed liquid of the issatchenkia orientalis to obtain a mixed bacterial liquid, wherein the pichia pastoris seed liquid accounts for 60-80% of the mixed bacterial liquid;
(5) tobacco fermentation: adding mixed bacteria solution into tobacco according to 0.4ml/g, supplementing sterile water 0.2ml/g tobacco every 3d, culturing at 28-30 deg.C for 15-20d,
drying at 40-60 deg.C for 15-30 min.
2. The method for fermenting tobacco by mixed bacteria according to claim 1, wherein: and (3) in the tobacco fermentation step in the step (5), the tobacco can be cut into threads before being fermented, and can also be fermented after being cut into threads to obtain a finished product.
3. The method for fermenting tobacco by using mixed bacteria as claimed in claim 1 or 2, wherein: the slant culture medium is malt extract agar culture medium, and comprises 130g/L malt extract powder, 0.1g/L chloramphenicol, 15g/L agar, and pH of 5.8-6.2.
4. The method for fermenting tobacco by using mixed bacteria as claimed in claim 3, wherein: pichia pastoris FBKL2.0008, accession number: CCTCC NO: m2015380.
5. The method for fermenting tobacco by using mixed bacteria as claimed in claim 3, wherein: issatchenkia orientalis FBKL2.0002 with the preservation number: CCTCC NO: m2018251.
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| CN110699342A (en) * | 2019-09-25 | 2020-01-17 | 内蒙古昆明卷烟有限责任公司 | Application of aspergillus oryzae LCCC30141 in production of neutral protease and tobacco leaf fermentation |
| CN110897193B (en) * | 2019-12-04 | 2022-06-17 | 广西中烟工业有限责任公司 | Preparation method of black malt extract and application of black malt extract in cigarettes |
| CN113846125B (en) * | 2021-09-06 | 2024-01-30 | 云南省烟草农业科学研究院 | Bacillus subtilis, phaffia rhodozyma and rhizopus mixed tobacco fermentation method |
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